When organs of the body are formed, they develop in neatly organized arrays. Often, cell groups of one kind are separated from cells of another kind by flat strips of connective tissue called basement membranes. In skin, for instance, the superficial layer of epidermal cells adheres to the underlying basement membrane. This skin basement membrane acts as a barrier between the epidermal cells on the outside, and the dermal cells underneath. A similar arrangement of cells occurs in the lining of the gut.
Basement membranes have been implicated in the growth, attachment, migration, repair, and differentiation of their overlying cell populations. Three layers have been defined in basement membranes: a) the Lamina lucida, an electronmicroscopically clear region that resides in close approximation to the overlying cells; b) the lamina densa, an electron dense region of 20-300 nm in width; and c) the sublamina densa that contains anchoring fibrils, microfibrillar bundles and collagen fibers.
Many different types of compounds have now been localized to the basement membrane. Some of these compounds are laminin, collagen IV and heparin sulfate proteoglycans (Verrando et al. Exp. Cell Res. (1987); 170: 116-128). In addition, specific basement membranes include other biologically active components, such as nidogen and entactin.
The principal cell adhesion receptor that epidermal cells use to attach to the basement membrane is called .alpha.6.beta.4. This transmembrane receptor is formed by a combination of two protein moieties .alpha.6 and .beta.4. The .alpha.6 and .beta.4 proteins are derived from different genes that have been found to be part of the integrin family.
Integrins are versatile family cell adhesion receptors. So far, approximately twenty members have been discovered in the integrin family. These molecules are involved in many types of cell adhesion phenomena in the body. Integrins are signalling molecules that can translate environmental cues into cellular instructions. Further, integrins can also transmit signals in the reverse direction, from the cell interior to the exterior. This has been illustrated in non-adherent cells, such as lymphocytes.
Stimulation of the T-cell antigen receptor, or of the CD3 complex, augments the affinity of certain integrins for their respective ligands. Unfortunately, in adherent cells, changes in the affinities of integrins have been more difficult to demonstrate. However, affinity modulation of one integrin in differentiating epidermal keratinocytes has been described by Adams et al. (Cell (1990); 63:425-435). For this reason, modifications of cell status initiated by activation or differentiation of other receptors may influence integrin affinity, and ultimately, the adhesive behavior of cells. Further, as a consequence of adhering to a surface, an integrin may actively contribute to modifying cell shape or migration.
Many epithelial cells interact with the underlying extracellular matrix via a junction called the hemidesmosome (Staehelin, 1974). Over the last few years there has been considerable progress in the biochemical characterization of this junction (Schwartz, et al., 1990). The hemidesmosome, with its associated structures such as intermediate filaments and anchoring fibrils, forms an adhesion complex. Disruptions of the epithelial-connective tissue interaction are often accompanied by disruption of the hemidesmosome complex. For example, in certain blistering skin diseases such as junctional epidermolysis bullosa where epithelial cell-connective tissue interaction is abnormal, it has been proposed that there is a biochemical modification in or loss of a basement membrane zone-associated component of the hemidesmosome.
Two high molecular weight intracellular components of the hemidesmosome have been identified and characterized with the aid of antisera from patients suffering from bullous pemphigoid. This autoimmune disease results in a disruption of the interactions between epithelial cells and connective tissue simultaneously with loss of hemidesmosome integrity (Chapman et al. Br. J. Dermatol (1990); 123:137-144). Accordingly, it was discovered that bullous pemphigoid patients were producing antibodies against hemidesmosome components. Two hemidesmosome related bullous pemphigoid (BP) antigens have been previously described (Klatte, et al., 1989).
One BP antigen is a 230 kD polypeptide that may act as an anchor for cytoskeleton elements in the hemidesmosomal plaque (Jones and Green, 1991). A second BP antigen is a type II membrane protein that possesses a collagen-like extracellular domain (Giudice, et al., 1991; Hopkinson, et al., 1992). In addition, it has been demonstrated that the interaction of the hemidesmosome with the underlying connective tissue involves the .alpha..sub.6 .beta..sub.4 integrin heterodimer (Stepp, et al., 1990; Jones, et al., 1991; Sonnenberg, et al., 1991; Kurpakus, et al., 1991). The .alpha..sub.6 .beta..sub.4 heterodimer has been localized to hemidesmosomes along the basal surfaces of the rat bladder carcinoma cell line 804G (Jones et al. Cell Regulation (1991); 2:427-438). These results suggested that integrins (e.g. .alpha..sub.6 .beta..sub.4) may play an important role in the assembly and adhesive functions of hemidesmosomes.
Various prior art efforts have focused on purifying adhesion-facilitating proteins found in basement membrane. For example, Burgeson, et al., Patent Cooperation Treaty Application No. WO92/17498, disclose a protein which they call kalinin. Kalinin is said to facilitate cell adhesion to substrates; however, this material is apparently inactive with respect to hemidesmosome formation. See also, Marinkovich, et al., J. Cell Biol. (1992); 119:695-703 (k-laminin); Rouselie, et al., J. Cell. Biol. (1991); 114:567-576 (kalinin); and Marinkovich, et al., J. Biol. Chem. (1992); 267:17900-17906 (kalinin).
Similarly, a basement glycoprotein of about 600 kD made up of polypeptides in the range of 93.5 kD to 150 kD has been identified, and is known as GB3 or nicein. See, e.g., Verrando, et al., Biochim. Biophys. Acta (1988); 942:45-56; and Hsi, et al., Placenta (1987); 8:209-217. None of these materials have been effective in generating formation of hemidesmosomes, either in vitro or in vivo.
When cultured on tissue culture plastic in vitro, most epithelial cells do not assemble bona fide hemidesmosomes despite the fact that they appear to express all of the hemidesmosomal plaque and transmembrane components mentioned above. Indeed, it is only recently that cell lines such as 804G were discovered to have the ability to readily assemble hemidesmosomes in vitro under regular culture conditions (Riddelle, et al., 1991; Hieda, et al., 1992). Such cells are at last allowing detailed cell and biochemical analysis of the dynamics of hemidesmosome assembly.
For instance, it has been reported that substratum-associated staining by anti-hemidesmosome antibodies is greatly diminished in 804G cell cultures that enter in vitro wound sites (Riddelle et al., J. Cell Sci. (1992); 103:475-490). However, as closure of the wound became complete, anti-hemidesmosome staining along the substratum-attached surface was evident in the cells.
There are, however, many epithelial cells that do not attach to tissue culture dishes in a normal fashion, even after treatment with various growth factors. These cells do not produce normal hemidesmosomes or grow to resemble their in vivo phenotype. It would provide a tremendous advantage to have a system that was capable of maintaining epithelial cell growth in vitro wherein the cells maintained their normal phenotype.